Molecular characterization of goose- and chicken-type lysozymes in emu (Dromaius novaehollandiae): evidence for extremely low lysozyme levels in emu egg white

Lysozyme (LZ), a bacteriolytic enzyme, is found in the egg white of many avian eggs and plays an important role in host defense; however, LZ activity in emu (Dromaius novaehollandiae) egg white is exceptionally undetectable. We cloned and characterized emu goose-type LZ (LZG) and chicken-type LZ (LZC) genes. RT-PCR analysis revealed very low LZG gene expression levels and absence of LZC gene expression in the emu oviduct. Sequencing of full-length LZG and LZC cDNAs indicated that their amino acid sequences show high similarities to ostrich LZG and LZC, respectively, with conserved catalytic residues for enzymatic activities. Whereas recombinant emu LZG prepared using Pichia pastoris exhibited similar enzyme activity as ostrich LZG, recombinant emu LZC exhibited significantly higher lytic activity than chicken LZC. We concluded that emus have functional genes for both LZG and LZC like many other avians, and the LZG gene is expressed in oviduct probably as in other ratite, however, its expression levels in egg white were low to be detected.     

Haplotypes and effects on growth traits of bovine Wnt7a gene in Chinese Qinchuan cattle

Wnt7a is a member of the WNT gene family, which encodes secreted signaling proteins and responds to many biological processes. Specifically Wnt7a influences satellite stem cells and regulates the regenerative potential of the muscle. However, similar researches about the bovine Wnt7a gene are lacking. Therefore, in this study, polymorphisms of the bovine Wnt7a gene were detected in 488 individuals from Chinese Qinchuan cattle by DNA pooling, forced PCR-RFLP, and DNA sequencing methods. 3 novel SNPs were identified, two SNPs (g.T4926C and g.A21943G) were in the intron and the last one (g.C63777T) was in the exon. Five haplotypes involved in these three variant sites in the Wnt7a gene were identified and their effects on growth traits were analyzed. The results revealed that haplotype 1 had the highest haplotype frequencies and was highly significantly associated with body height (P < 0.01), body weight (P < 0.05), chest width (P < 0.05) and height at hip cross (P < 0.01) respectively.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Ontogenic expression pattern and genetic polymorphisms of the retinol-binding protein 4 (RBP4) gene in Erlang mountainous chickens

Retinol-binding protein 4 (RBP4) is the only circulatory transport protein for vitamin A. Based on the essential role of vitamin A in chicken reproduction, we measured RBP4 mRNA abundance in Erlang mountainous chickens. We also identified and analyzed the gene polymorphism and its effect on reproduction traits among 349 chickens. The expression of RBP4 mRNA showed specific developmental changes and striking differences among tissues. The mRNA abundance was greatest (P < 0.05) in the liver, intermediate in the ovary, kidney, small intestine, oviduct and heart, and lowest in the hypothalamus and pituitary, as compared to all other tissues (P < 0.05). We detected one single nucleotide polymorphism (g.19942455C>G) in intron 2 of the RBP4 gene. Three genotypes (CC, CG and GG) were identified, with a significant effect of genotype on the age at first egg (AFE), first egg weight (FEW), total eggs at 300 days (TE300), highest continuous laying days (HCLD) and average laying interval (ALI). The GG genotype, where chickens display earlier AFE, more TE300, longer HCLD and shorter ALI, would be genetically advantageous and its selection may improve reproduction traits. These results suggested that the RBP4 gene might play an important role in reproduction traits in chickens.     

Single nucleotide polymorphism scanning and expression of the FRZB gene in pig populations

Secreted frizzled-related protein 3 (sFRP3), encoded by the gene FRZB, is a member of the sFRP family with important roles in inhibition of the Wnt signalling pathway through competitive binding of the Wnt receptor. Here, we investigated pig FRZB as a candidate gene for growth traits and identified three polymorphic sites, an insertion (A-532B) and two SNPs (G636A and C650T) in its 5′-UTR. The genotype distributions of G636A and C650T were significantly different among mini-type indigenous (Diannan Small-ear and Tibetan), normal indigenous (Laiwu and Huai), and introduced (Large Yorkshire and Landrace) breeds. In semi-quantitative PCR expression analysis, expression of FRZB mRNA was abundant in tissues of hypophysis, longissimus dorsi muscle, and adipose tissues, and low in the heart, hypothalamus, and brain. Quantitative determination of mRNA level and protein expression analysis were corresponding. The results demonstrated that FRZB gene expression in longissimus dorsi muscle and liver tissue was significantly higher in Diannan Small-ear and Tibetan pigs than in the Large Yorkshire breed (P < 0.05); however, in back fat tissue, the expression was significantly higher in Diannan Small-ear pig than in Tibetan or Large Yorkshire breeds (P < 0.05). Given the known growth and fat characteristics of the breeds, these results indicate that FRZB expression has a negative association with muscle growth and a positive association with fat deposition. In conclusion, FRZB may be a major candidate gene for growth traits in pigs.     

Regulation of carboxylesterase 1 (CES1) in human adipose tissue

Carboxylesterase 1 (CES1) has recently been suggested to play a role in lipolysis. Our aim was to study the regulation of CES1 expression in human adipose tissue. In the SOS Sib Pair Study, CES1 expression was higher in obese compared with lean sisters (n = 78 pairs, P = 8.7 × 10⁻¹⁸) and brothers (n = 12 pairs, P = 0.048). CES1 expression was higher in subcutaneous compared with omental adipose tissue in lean (P = 0.027) and obese subjects (P = 0.00036), and reduced during diet-induced weight loss (n = 24, weeks 8, 16, and 18 compared to baseline, P < 0.0001 for all time points). CES1 expression was higher in isolated adipocytes compared with intact adipose tissue (P = 0.0018) and higher in large compared with small adipocytes (P = 4.1 × 10⁻⁶). Basal and stimulated lipolysis was not different in individuals with high, intermediate, and low expression of CES1. Thus, CES1 expression was linked to body fat and adipocyte fat content but not to lipolytic activity.     

Expression of zebrafish anterior gradient 2 in the semicircular canals and supporting cells of otic vesicle sensory patches is regulated by Sox10

AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(− 6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(− 6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5′-end deletion revealed that a 100 bp sequence within the − 2.6 to − 2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the − 2.6 to − 2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.     

Hypoxia during pregnancy in rats leads to the changes of the cerebral white matter in adult offspring

The aim of the present study is to evaluate the effect of reduced fetal oxygen supply on cerebral white matter in the adult offspring and further assess its susceptibility to postnatal hypoxia and high-fat diet. Based on a 3 × 2 full factorial design consisting of three factors of maternal hypoxia, postnatal high-fat diet, and postnatal hypoxia, the ultrastructure of myelin, axon and capillaries were observed, and the expression of myelin basic protein (MBP), neurofilament-H+L(NF-H+L), and glial fibrillary acidic protein (GFAP) was analyzed in periventricular white matter of 16-month-old offspring. Demyelination, injured axon and damaged microvasculars were observed in maternal hypoxia offspring. The main effect of maternal hypoxia lead to decreased expression of MBP or NF-H+L, and increased expression of GFAP (all P < 0.05). Moreover, there was positive three-way interaction among maternal hypoxia, high-fat diet and postnatal hypoxia on MBP, NF-H+L or GFAP expression (all P < 0.05). In summary, our results indicated that maternal hypoxia during pregnancy in rats lead to changes of periventricular white matter in adult offspring, including demyelination, damaged axon and proliferated astroglia. This effect was amplified by high-fat diet and postnatal hypoxia.     

Landscape of the relationship between type 2 diabetes and coronary heart disease through an integrated gene network analysis

Type 2 diabetes (T2D) and coronary artery disease (CAD) are closely related chronic diseases with high prevalence and morbidity. However, a comprehensive comparison of the two diseases is lacking. Recent genome-wide association studies (GWAS) have identified a handful of single nucleotide polymorphisms (SNPs) that are significantly associated with the risk of T2D and CAD. These most significant findings may help interpret the pathogenesis of T2D and CAD. However, tremendous results from these GWAS are ignored. Here we revisited the raw datasets of these GWAS and performed an integrated gene network analysis to unveil the relationship between T2D and CAD by combining multiple datasets including protein–protein interaction (PPI) database, publication libraries, and pathway datasets. Our results showed that majority of genes were involved in the first module (1122 genes in T2D and 895 in CAD). Four pathways were found to be common in both T2D and CAD, including regulation of actin cytoskeleton, calcium signaling pathway, MAPK signaling pathway and focal adhesion (all P < 0.00001). MAX which was involved in small cell lung cancer pathway was a hub gene unique to T2D (OR = 1.2, P = 0.006) but not in CAD. In contrast, three hub genes including PLEKHG5 (T2D: OR = 1, P = 1; CAD: OR = 1.12, P = 0.006), TIAM1 (T2D: OR = 1, P = 1; CAD: OR = 1.48, P = 0.004) and AKAP13 (T2D: OR = 1, P = 1; CAD: OR = 1.38, P = 0.001) were hub genes unique to CAD. Moreover, for some hub genes (such as SMAD3) that were susceptible to both T2D and CAD, their associated polymorphisms were unique to each of the two diseases. Our findings might provide a landscape of the relationship between T2D and CAD.     

Association of A-FABP gene polymorphism in intron 1 with meat quality traits in Junmu No. 1 white swine

This study was designed to investigate the single nucleotide polymorphism (SNP) in intron 1 of the gene A-FABP in 127 Junmu No. 1 white swine using PCR-SSCP. The association between the polymorphism and meat quality traits was also studied. The cloning and sequencing results indicated that the polymorphism of intron 1 was due to a point mutation in position 3481 bp of A-FABP, giving 3 genotypes (CC, CD and DD). Association analysis indicated that the polymorphism had a significant effect on marbling (P < 0.05). Genotype DD had higher marbling than CD and CC, but the difference between CD and CC was no significant. Polymorphism had a highly significant effect on intramuscular fat (IMF) content (P < 0.01). DD was higher than CD, which was higher than CC. No significant conclusions can be drawn regarding other traits. Immunoblot analysis of A-FABP levels was carried out on 3 different genotype individuals. Expression was markedly reduced in DD compared with genotype CC. Thus A-FABP may be a candidate gene or a quantitative trait locus-linked gene associated with meat quality traits.     

The relationship between five widely-evaluated variants in CDKN2A/B and CDKAL1 genes and the risk of type 2 diabetes: A meta-analysis

The genes encoding two cyclin-dependent kinases-inhibitor-2A/B (CDKN2A/B) and 5 regulatory subunit-associated protein-like 1 (CDKAL1) have been investigated extensively in associations with type 2 diabetes; the results, however, are often irreproducible. We therefore sought to evaluate these associations by performing a meta-analysis on five widely-evaluated variants from the two genes. There were 38 studies (patients/controls: 51,940/52,234) for rs10811661, 16 studies (20,029/24,419) for rs564398 in CDKN2A/B gene, and 27 studies (28,383/47,635) for rs7756992, 26 studies (28,816/31,713) for rs7754840, 21 studies (29,260/38,400) for rs10946398 in CDKAL1 gene. Overall risk estimates for type 2 diabetes conferred by rs10811661-T, rs564398-A, rs7754840-C, rs7756992-G, and rs10946398-C alleles were 1.17 (95% CI: 1.10–1.23; P < 0.0005; I² = 83.9%), 1.1 (95% CI: 1.0–1.21; P = 0.051; I² = 88.3%), 1.24 (95% CI: 1.18–1.3; P < 0.0005; I² = 74.3%), 1.2 (95% CI: 1.11–1.3; P < 0.0005; I² = 92.0%), and 1.19 (95% CI: 1.1–1.29; P < 0.0005; I² = 90.8%), respectively. There was evident publication bias for rs564398 and rs7754840. Subgroup analyses by ethnicity showed remarkable divergences in risk estimate for rs564398 between Asians (odds ratio [OR] = 1.01; 95% CI: 0.86–1.19; P = 0.868) and Caucasians (OR = 1.19; 95% CI: 1.03–1.35; P = 0.012) (P < 0.05). For all variants examined, the results of studies in retrospective design or with population-based controls were comparative with that of overall studies. In meta-regression analyses, age was found to exert a significant influence on the association between rs10811661 and type 2 diabetes (P = 0.003), as well as between rs7754840 and gender (P = 0.034). Taken together, our findings provide evidence for a significant contribution of CDKN2A/B gene rs10811661 and CDKAL1 gene rs7756992 and rs10946398 to type 2 diabetes.     

Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6

Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K_i around 1 nM, KLK4 with K_i = 27.3 nM, KLK6 with K_i = 140 nM, caspase-14 with a K_i approximating 1 μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members.     

The MTMR9 rs2293855 polymorphism is associated with glucose tolerance,insulin secretion,insulin sensitivity and increased risk of prediabetes

Background

Polymorphism of rs2293855 in gene MTMR9 has been associated with obesity and metabolic syndrome. We aim to study the association of rs2293855 with type 2 diabetes mellitus (T2DM) intermediate phenotypes in a Han Chinese population.

Methods

The polymorphism was genotyped in 838 Han Chinese individuals using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS); all participants underwent a 75 g oral glucose tolerance test (OGTT); associations between the polymorphism and glucose tolerance, indices of insulin secretion and indices of insulin sensitivity were analyzed.

Results

The frequency of genotypes and alleles differed significantly between normal glucose tolerance and prediabetes (P = 0.043 and P = 0.009, respectively). The GG homozygous presented higher fasting plasma glucose (P = 0.009), higher 2-hour plasma glucose (P = 0.024) and higher glucose area under the curve (AUC, P = 0.01). Moreover, the G allele of rs2293855 was associated with glucose intolerance (fasting glucose, P = 0.012; glucose AUC, P = 0.006; 2-h glucose, P = 0.024); it is also associated with decreased indices of insulin sensitivity (fasting insulin, P = 0.043; insulin sensitivity index composite, P = 0.009; homeostasis model assessment of insulin resistance, HOMA-IR, P = 0.008) and decreased indices of insulin secretion (HOMA of beta cell function, HOMA-B, P = 0.028; insulinogenic index, P = 0.003). In addition, the minor allele G was also associated with increased risk of prediabetes (OR = 1.463, 95%CI: 1.066–2.009, P = 0.018).

Conclusions

Polymorphism of rs2293855 in MTMR9 is associated with measures of glucose tolerance, indices of insulin secretion and indices of insulin sensitivity. We also suggest that allele G is likely to increase the risk of prediabetes by influencing both insulin secretion and insulin sensitivity.     

Effects of supplemental levels of hesperetin and naringenin on egg quality, serum traits and antioxidant activity of laying hens

Hesperetin and naringenin phytochemicals are naturally occurring flavanoids in citrus fruits. The purpose of this study was to evaluate the effects of supplementing different levels of extracted hesperetin and naringenin on egg quality, serum traits and antioxidant activity in laying hens. Two experiments were conducted, each for 10 weeks, in a completely randomized experiment design. Each had 100 Leghorn laying hens (26 weeks old) randomly assigned into five groups (n = 20) based on dietary categories of hesperetin 0, 0.5, 1, 2, 4 g/kg and naringenin 0, 0.5, 1, 2, 4 g/kg. Experimental results indicated that there was increased (P<0.05) egg production in the 1 g/kg naringenin-supplemented group, but lower (P<0.05) egg production in the hesperetin- and naringenin-supplemented groups given 4 g/kg. Cholesterol content (per gram yolk) and total cholesterol content (per egg) were lower (P<0.05) in the hesperetin- and naringenin-supplemented groups as compared to the control group, and the 2 g/kg hesperetin- and naringenin-supplemented groups showed the most significant difference. Both serum cholesterol and triglyceride concentrations were lower (P<0.05) in the 2 g/kg hesperetin- and naringenin-supplemented groups. The SOD and catalase activities, scavenging O₂⁻ and iron-chelating abilities were higher (P<0.05) in the 2 g/kg hesperetin- and naringenin-supplemented groups, and the trolox equivalent antioxidant capacity was higher (P<0.05) in the 2 g/kg naringenin-supplemented group. The results confirmed that both hesperetin and naringenin could lower serum and egg yolk cholesterol levels, and improve the antioxidant activities, however the measured variables generally showed significant quadratic responses to increasing amounts of the compounds. The recommended supplementation level of hesperetin and naringenin is 2 g/kg of the basal diet for reduced serum and yolk cholesterols contents and increased antioxidant capacity.